The broad and long-term goal of this application is to identify the molecular mechanisms that cause facioscapulofaciohumeral dystrophy (FSHD). The broad hypothesis is that deletion of a subset of the D4Z4 units on 4qA161 results in increased or aberrant transcription from the remaining D4Z4 units on the deleted allele. Our preliminary studies have identified multiple sense and anti-sense transcripts from the D4Z4 unit. We have characterized one of the polyadenylated D4Z4 sense transcripts and developed assays to demonstrate its biological function. The specific goal of this application is to determine whether the protein product of a D4Z4 transcript causes FSHD. Aim 1 will characterize sense and anti-sense transcripts from pathogenic and non-pathogenic D4Z4 alleles, their potential protein products, and their expression relative to FSHD. Aim 2 will test the hypothesis that the polyadenylated RNA transcripts from the D4Z4 locus encode several distinct proteins, including the full-length DUX4 and splice-isoforms of DUX4, each with specific developmental biological functions in myogenesis, and that these functions contribute to FSHD pathology. Aim 3 will characterize the IRES element of the DUX4 RNA and test a new hypothesis regarding transitions between RNA translational programs during myogenic differentiation. Together, these studies will advance our knowledge regarding the pathophysiology of FSHD and muscle cell biology.